A parabrachial to hypothalamic pathway mediates defensive behavior

Defensive behaviors are critical for animal’s survival. Both the paraventricular nucleus of the hypothalamus (PVN) and the parabrachial nucleus (PBN) have been shown to be involved in defensive behaviors. However, whether there are direct connections between them to mediate defensive behaviors remains unclear. Here, by retrograde and anterograde tracing, we uncover that cholecystokinin (CCK)-expressing neurons in the lateral PBN (LPBCCK) directly project to the PVN. By in vivo fiber photometry recording, we find that LPBCCK neurons actively respond to various threat stimuli. Selective photoactivation of LPBCCK neurons promotes aversion and defensive behaviors. Conversely, photoinhibition of LPBCCK neurons attenuates rat or looming stimuli-induced flight responses. Optogenetic activation of LPBCCK axon terminals within the PVN or PVN glutamatergic neurons promotes defensive behaviors. Whereas chemogenetic and pharmacological inhibition of local PVN neurons prevent LPBCCK-PVN pathway activation-driven flight responses. These data suggest that LPBCCK neurons recruit downstream PVN neurons to actively engage in flight responses. Our study identifies a previously unrecognized role for the LPBCCK-PVN pathway in controlling defensive behaviors.


Introduction
Living in an environment full of diverse threats, animals develop a variety of defensive behaviors for survival during evolution (Anderson and Adolphs, 2014;Blanchard et al., 2001;Crawford and Masterson, 1982;Fanselow and Bolles, 1979;LeDoux, 2012;Yilmaz and Meister, 2013). From the perspective of predator and prey interactions, defensive responses are generally divided into two categories: to avoid being discovered (such as freezing or hiding behaviors) or being caught (such as escaping or attacking behaviors) (Crawford and Masterson, 1982;Fanselow and Bolles, 1979;Yilmaz and Meister, 2013). Which type of defensive behavior is selected depends on many factors, including the intensity and proximity of the threat, as well as the surrounding context. In general, escape is triggered when animals are faced with an imminent threat with a shelter nearby (Eilam, 2005;Lin et al., 2023;Perusini and Fanselow, 2015;Sun et al., 2020b;Zhang et al., 2018). Several brain regions, including the periaqueductal gray (PAG), amygdala, and medial hypothalamic zone (MHZ), have been implicated in mediating escape behaviors (Evans et al., 2018;Shang et al., 2018;Shang et al., 2015;Silva et al., 2013;Tovote et al., 2016;Wang et al., 2015;Wang et al., 2021b;Wei et al., 2015). Recent studies have highlighted a role for the hypothalamus, in particular the MHZ, including the anterior hypothalamic nucleus (AHN), the dorsomedial part of ventromedial hypothalamic nucleus (VMHdm) and the dorsal pre-mammillary nucleus (PMd), in mediating escape behaviors. For example, a subset of VMH neurons collaterally project to the AHN and PAG to promote both escape and avoidance behaviors . Moreover, PMd neurons project to the dorsolateral periaqueductal gray region (dlPAG) and the anteromedial ventral thalamic region (AMV) also control escape behaviors (Wang et al., 2021b). In addition, PVN, an area enriched with endocrine neurons, has also been associated with escape behaviors . Optogenetic activation of Sim1 + neurons in the PVN triggers escape and projections from the PVN to both the ventral midbrain region (vMB) and the ventral lateral septum (LSv) mediate defensive-like behaviors, including hiding, escape jumping and hyperlocomotion Xu et al., 2019). Despite a growing number of studies illustrating the importance of PVN in mediating defensive behaviors, upstream inputs that transmit the danger signals to the PVN remain unclear (Isosaka et al., 2015;Penzo et al., 2015).
Located in the dorsolateral pons, PBN, serves as an important relay station for sensory transmission (Fulwiler and Saper, 1984). While PBN is best known for various sensory processes to protect the body from noxious stimuli, emerging evidence suggests that it may act as a key node in mediating defensive behaviors (Campos et al., 2018;Day et al., 2004;Han et al., 2015a). Electrical lesions of the PBN or local microinjections of kainic acid into the PBNinduced defensive behaviors (Mileikovskii and Verevkina, 1991). In addition, exposure of the olfactory predator cue, trimethylthiazoline (TMT), increased c-fos expression in the LPB (Day et al., 2004). More importantly, calcitonin gene-related peptide (CGRP) neurons, located in the external lateral subnuclei of PBN (PBel), have been shown to mediate alarm responses and defensive behaviors under stressful or threatening circumstances via projections to the amygdala and the bed nucleus of stria terminalis (BNST) ( Han et al., 2015a;Zhang et al., 2020). While both PBN and PVN neurons appear to be involved in defensive responses, it remains unclear whether they connect with each other to mediate defensive behaviors (Fulwiler and Saper, 1984).
Both CCK and CCK receptor-expressing neurons have been implicated in defensive behaviors (Bertoglio et al., 2007;Chen et al., 2022;Wang et al., 2021a). PVN neurons also express CCK receptors and are involved in defensive behaviors O'Shea and Gundlach, 1993;Xu et al., 2019), raising a possibility that upstream CCK inputs to the PVN (Meister et al., 1994) may be implicated in defensive behaviors. In an attempt to test this possibility, we injected the Cre-dependent retrograde tracer (AAV2-Retro-DIO-EYFP) into the PVN of Cck-cre mice and found that the LPB is an important upstream CCKergic input to the PVN. We further showed that LPB CCK neurons were recruited upon exposure to various threat stimuli. Optogenetic activation of either LPB CCK neuronal somas or their projections to the PVN promotes defensive responses, whereas their inhibition attenuates defensive-like behaviors, suggesting an essential and sufficient role for LPB C-CK -PVN pathway in mediating defensive behaviors.

LPB CCK neurons provide monosynaptic glutamatergic projections to the PVN
To identify upstream CCKergic inputs to the PVN and visualize the range of viral infection, we injected a mixture of retrograde viral tracer (AAV2-Retro-DIO-EYFP, hereafter referred to as AAV2-Retro) and Wang, Chen et  CTB 555 into the PVN of knock-in mice expressing Cre recombinase at the CCK Locus (Cck-ires-cre, referred to as Cck-cre hereafter; Figure 1A). We observed abundant EYFP + neurons in the LPB, medial orbital cortex (MO), and cingulate cortex (CC), with scattered EYFP + cells in the PAG and dysgranular insular cortex (DI) ( Figure 1B-H). Since LPB is a region critical for sensory signal processing, we mainly focused on the LPB in the following study (Fulwiler and Saper, 1984;Garfield et al., 2014). LPB contains several subregions with diverse peptide-expressing neuronal subtypes and CCK + neurons have been shown to primarily locate within the superior lateral PB (PBsl; Garfield et al., 2014). To dissect the identity of CCK + neurons in the LPB, we performed in situ hybridization in Cck-cre::Ai14 mice using probes for Slc17a6 and Slc32a1, markers specific to glutamatergic and GABAergic neurons (Figure 1-figure supplement 1A-C). We found that LPB CCK neurons (84.0% ± 1.7%) predominantly expressed Slc17a6, with a tiny subpopulation (2.0% ± 1.2%) expressing Slc32a1, suggesting that LPB CCK neurons were mostly glutamatergic neurons (Figure 1-figure supplement 1D-E). In addition, co-labeling with another neuropeptide, CGRP, revealed minimal co-localization (2.2% ± 0.37%) between CCK and CGRP ( Figure 1-figure supplement 1F-G), suggesting primary separation between these two subpopulations of neurons. Collectively, these data demonstrate that LPB CCK neurons comprise predominantly glutamatergic neurons, which barely overlap with CGRP neurons.
Photostimulation of LPB CCK neurons induces aversion and defensive behaviors LPB CCK neurons have been shown to regulate glucose homeostasis and body temperature (Garfield et al., 2014;Yang et al., 2020). However, it remains unclear whether activation of LPB CCK neurons may elicit direct behavioral changes. Before optic stimulation in vivo, we first tested the efficacy of optogenetic stimulation by whole-cell recording in ChR2-expressing LPB CCK neurons. We observed that 5-20 Hz blue laser pulses induced time-locked action potential firing, with 20 Hz inducing maximal firing capacities. We then chose 20 Hz for the following in vivo stimulation ( Figure 2A).
We injected the control or ChR2-expressing viruses (AAV2/9-EF1a-DIO-EYFP or AAV2/9-EF1a-DIO-ChR2-EYFP) into the LPB of Cck-cre mice, followed by optical fiber implantation and optic stimulation ( Figure 2B-D). Since LPB has been shown to mediate aversion (Chiang et al., 2019), we first tested whether optogenetic activation of LPB CCK neurons affects aversion using the real-time place aversion (RTPA) test ( Figure 2E). We found that activation of LPB CCK neurons significantly reduced the duration of mice in the laser-paired chamber, accompanied by rapid running or flight to the other laserunpaired chamber, indicating an obvious aversion-like avoidance and/or fear-related defensive-like behaviors ( Figure 2F-G).
To further investigate whether LPB CCK neurons plays a role in defensive behaviors, we used a wellestablished flight-to-nest behavioral test by putting a nest in the corner of an arena to test the ability of mice to actively search for hiding ( Figure 2H). Of note, activating LPB CCK neurons induced robust flight-to-nest behavior in mice, with much shorter latency and faster speed running towards the nest (latency: EYFP, 87.71 ± 22.38 s vs. ChR2, 7.571 ± 0.8123 s; speed: EYFP, 184.4% ± 30.4% vs. ChR2, 361.7% ± 31.5%), followed by longer stay in the nest (EYFP, 63.906% ± 3.645% vs. ChR2, 97.26% ± 2.737%; Figure 2I-K). These data suggest that activation of LPB CCK neurons induces aversion-like avoidance and defensive-like flight-to-nest behaviors.  Defensive behaviors are usually accompanied by changes in the autonomic nervous system, such as increased heart rates, dilated pupil size and elevated cortisol levels (Dong et al., 2019;Gross and Canteras, 2012;Wang et al., 2015). We found that photostimulation of LPB CCK neurons also significantly increased heart rates and enlarged pupil diameters, along with elevated plasma levels of corticosterone ( Figure 2L-P), suggesting that activating LPB CCK neurons induces fast sympathetic changes, leading to an arousal state accompanying the defensive responses in mice (Salay et al., 2018).
Since activation of LPB CCK neurons induced fear-associated flight-to-nest behaviors and long-term fear may trigger anxiety-like states in animals (Tovote et al., 2005;Yang et al., 2016), we also examined whether prolonged activation of LPB CCK neurons induces anxiety-like behaviors ( LPB CCK neurons encode threat stimuli-evoked flight behaviors LPB neurons were shown to be activated when animals are in proximity to dangerous stimuli (Campos et al., 2018;Day et al., 2004;Han et al., 2015b). To track the endogenous activities of LPB CCK neurons in vivo, we injected the Cre-dependent fluorescent calcium indicator AAV-DIO-GCaMP7s into the LPB of Cck-cre mice, and recorded the calcium responses by fiber photometry ( Figure 3A-C). Consistent with previous observation (Yang et al., 2020), we observed elevated calcium responses of LPB CCK neurons by heat stimuli (43 °C) (Figure 3-figure supplement 1A-D). We then tested the response of these neurons in a predator-exposure assay, in which an awake but restrained rat was placed at one end of a rectangular arena (Reis et al., 2021;Weisheng et al., 2021), and a mouse was placed at the other end, away from the rat. After perceiving the presence of the rat, mice usually exhibit risk assessment behaviors by curiously approaching and investigating the rat (Olivier et al., 1991). We observed that the calcium signals of LPB CCK neurons gradually rise when mice approached the rat. At the moment of escape initiation, when mice turned back and dashed away from the rat, the calcium signals reached a peak ( Figure 3D-G and L). However, the signals ramped down as mice gained distance from the rat. Notably, activities of LPB CCK neurons were unaffected in the presence of a toy rat (Figure 3-figure supplement 1E-H).
In the presence of the visual predatory cue, such as appearing and expanding looming shadows to mimic an approaching predator (Yilmaz and Meister, 2013), LPB CCK neurons also showed increased calcium signals ( Figure 3H). Similar to rat exposure assay, we also tested the response of these neurons in the looming assay, the elevated calcium signals reached peak when the animal initiated escape, but reduced once it ran into the nest ( Figure 3H-K and M). LPB CCK neurons also showed elevated calcium signals when mice were exposed to the olfactory predatory cue, TMT odor (

Inhibition of LPB CCK neurons suppressed predatory cue-evoked flight responses
To examine whether LPB CCK neurons are required for innate threat-evoked defensive behaviors, we then inhibited LPB CCK neurons using optogenetic tools. By injecting the Cre-dependent AAV expressing the guillardia theta anion channel rhodopsins-1 (GtACR1) into the LPB of Cck-cre mice, followed by optic stimulation, we were able to effectively inhibit the firing of LPB CCK neurons ( Figure 4A). Next, we bilaterally injected the control or GtACR1 viruses into the LPB and photo-inhibited these neurons in vivo ( Figure 4B-C). In the rat exposure test ( Figure 4D-E), after a quick exploration of the rat in the corner (the danger zone), control mice usually fled to the other end of the box (the putative safe zone). However, optic inhibition of LPB CCK neurons significantly increased the time (EYFP, 7.247% ± 2.329% vs. GtACR1, 26.57% ± 5.375%) and entries (EYFP, 6.167 ± 1.579 vs. GtACR1, 12.67 ± 2.141) of mice to the danger zone, with no effect on total travel distance (EYFP, 8.837 ± 1.934 m vs. GtACR1, 12.69 ± 1.421 m), suggesting a delayed flight response ( Figure 4F-H). In the looming test ( Figure 4I-J), inhibition of LPB CCK neurons also increased the latency fleeing to the nest (EYFP, 10.18 ± 2.828 s vs. GtACR1, 27.14 ± 6.526 s), followed by reduced hiding time in the nest (EYFP, 85.36% ± 9.846% vs. GtACR1, 39.17% ± 16.36%; Figure 4K-L). Our data suggest that LPB CCK neurons are required for proper defensive behaviors to rat exposure and looming stimuli.

Stimulation of the LPB CCK -PVN pathway triggers defensive-like flightto-nest behaviors
To further investigate whether activation of the LPB CCK -PVN pathway induces defensive-like flightto-nest behavior, we unilaterally injected the control or ChR2 virus into the LPB of Cck-cre mice and implanted an optical fiber above the PVN ( Figure 5A-E). By photostimulating the axonal terminals of LPB CCK neurons within the PVN, we also observed shorter latency (EYFP, 73.43 ± 21.08 s vs. ChR2, 8 ± 1.047 s), faster speed (EYFP, 122.3% ± 17.81% vs. ChR2, 277.9% ± 32.85%) running towards the nest, and longer stay in the nest, (EYFP, 3.751% ± 1.23% vs. ChR2, 89.29% ± 10.71%; Figure 5F-H), similar to soma activation. Post-hoc c-fos staining further verified the activation of PVN neurons after optic stimulation ( Figure 5I-J). As well, terminal activation also increased heart rates ( Figure 5K) and plasma corticosterone levels ( Figure 5N), but with no effects on pupil size ( Figure       these data suggest that activation of the LPB CCK -PVN pathway is sufficient to trigger flight-to-nest behavior, along with increased heart rates and corticosterone levels.

PVN is required for defensive responses to threatening situations
Since LPB CCK neurons project to multiple regions ( Figure 1-figure supplement 2A-I), the effects observed upon their activation may arise from different downstream targets, other than the PVN. To test this possibility, we investigated whether inhibition of the PVN is required for the LPB CCK neuronsinduced defensive-like flight-to-nest behavior. To achieve this aim, we used a dual virus-mediated optogenetic activation and chemogenetic inhibition strategy, by injecting AAV2/9-DIO-ChR2-EYFP into the LPB, and AAV2/9-hsyn-hM4Di-mCherry into the PVN of Cck-cre mice. By putting an optical fiber above the PVN, we were able to activate the LPB CCK terminals and induce defensive-like flight-tonest behavior as shown above ( Figure 6A-B). Upon CNO administration to inhibit PVN neurons, we observed a much longer latency, lower speed of the mice in returning to the nest, and reduced hiding time in the nest (latency: saline, 8.286 ± 1.475 s vs. CNO, 18.29 ± 2.843 s; speed: saline, 289.3% ± 25.22% vs. CNO, 124.7% ± 13.67%; time in the nest: saline, 81.55% ± 11.16% vs. CNO, 25.83% ± 10.32%; Figure 6C-E). These data suggest that the PVN is a required downstream target for LPB CCK neurons activation-induced defensive behavior.
LPB CCK neurons may release either glutamate or neuropeptide CCK to induce the defensive behavior. To determine which neurotransmitter or modulator is engaged in the above responses, we combined pharmacologic with optogenetic manipulation. Two types of CCK receptors are present in the central nervous system and Cckar is widely distributed throughout the PVN, whereas the expression of Cckbr in PVN is low ( Figure 6-figure supplement 1A-B). We infused glutamate receptor antagonists (CNQX +AP5), CCK receptor antagonists (Devazepide +L-365,260), or artificial cerebrospinal fluid (ACSF) through an implanted cannula 30 min before optogenetic activation of LPB CCK neurons ( Figure 6F-H). We found that blocking glutamate receptors induced a longer latency, slower speed in returning to the nest, along with reduced hiding time in the nest ( Figure 6I-K), when compared with ACSF-treated mice. However, blocking CCK receptors showed no significant effects ( Figure 6I-K). These results suggest that glutamatergic transmission from LPB CCK neurons to the PVN mediates defensive-like flight-to-nest behavior.

Discussion
Defensive behaviors are actions that are naturally selected to avoid or reduce potential harm for the survival of animal species. While emerging evidence has suggested that LPB are associated with sensation of danger signals and aversive stimuli (Campos et al., 2018;Han et al., 2015b), we found that LPB CCK neurons were actively recruited upon exposure to various predatory stimuli. Activating LPB CCK neurons triggers aversive-like avoidance and defensive-like flight responses, whereas inhibition of these neurons suppressed predatory stimuli-induced defensive behaviors. Inhibition of downstream PVN neurons attenuated photoactivation of LPB CCK -PVN terminals-promoted flight responses, suggesting that LPB CCK -PVN pathway is important for the regulation of defensive responses. Our study thus reveals a new connection from the brainstem to the hypothalamus to regulate innate defensive behaviors ( Figure 6Q).
Hippocampal and cortical CCK-expressing neurons have been thought of mainly GABAergic Sun et al., 2020a;Whissell et al., 2015). We found that LPB CCK neurons are predominantly glutamatergic, similar to those in the amygdala . While the roles of LPB CGRP neurons have been associated with the passive defensive responses (Han et al., 2015b;Keay and Bandler, 2001), we show that activation of LPB CCK neurons primarily triggers active defensive responses, such as flight-to-nest behavior (Keay and Bandler, 2001). The fact that CGRP and CCK neurons are spatially segregated, responsive to distinct threat signals and involved in different defensive responses, indicating that LPB acts as an important integration center or hub to gate defensive responses when animals encounter different threats Tokita et al., 2009). It would be interesting to investigate whether LPB CCK and LPB CGRP neurons coordinately regulate defensive responses under different threatening situations.
In vivo calcium imaging showed that LPB CCK neurons increase firing upon exposure to predators and/or predatory cues, which occurs before the initiation of an escape behavior. These findings indicate that LPB CCK neurons may produce a preparatory signal before the escape initiation, which allows the downstream targets to further assess the threat signals, plan escape routes and take appropriate motor actions. LPB CCK neurons are well positioned to play such an important role in linking the imminent threat to escape initiation, as they receive inputs from both the peripheral and visceral sensory system and project to many brain regions involved in defensive responses including the hypothalamus, PVT and PAG (Cooper and Blumstein, 2015;Ellard and Eller, 2009;Han et al., 2015b;Krout and Loewy, 2000;Saper and Loewy, 1980;Sun et al., 2020b;Tokita et al., 2009). While PVN CRH or PAG CCK neurons have also been shown to be recruited during flight, their peak activity did not match the flight initiation, but occurred during flight (Daviu et al., 2020;La-Vu et al., 2022). The peak of the experimental apparatus with a nest in the corner. (F-H) Optogenetic activation of the LPB CCK -PVN pathway shortened the latency but increased the speed of animals towards the nest, with increased hiding time in the nest (EYFP: n = 7 mice, ChR2: n = 7 mice; for latency, p = 0.0012, U = 0; Mann-Whitney test; for speed, p = 0.0013, t = 4.163, df = 12; unpaired t test; for time in the nest, p = 0.0006, U = 0; Mann-Whitney test). (I-J) C-fos staining in the PVN (I) and quantification of c-fos positive cells in the PVN (J). Scale bar: 100 μm. (EYFP: n = 3 mice, ChR2: n = 3 mice; p = 0.0033, t = 6.253, df = 4; unpaired t test). (K) Mean heart rate analyses in EYFP and ChR2 groups (EYFP: n = 7 mice, ChR2: n = 7 mice; p = 0.0002, t = 5.249, df = 12; unpaired t test). (L) Example image of computerdetected pupil size before and during photoactivation of LPB CCK neurons. (M) Relative pupil size of animals (during/ before photostimulation of LPB CCK neurons) (EYFP: n = 6 mice, ChR2: n = 6 mice; p = 0.0022, U = 0; Mann-Whitney test). (N) Plasma corticosterone levels in EYFP and ChR2 groups (EYFP: n = 5 mice, ChR2: n = 5 mice; p = 0.0079, U = 0; Mann-Whitney test).
The online version of this article includes the following source data and figure supplement(s) for figure 5: Source data 1. Quantification of the flight-to-nest behavior and autonomic responses upon activation of LPB CCK-PVN pathway.   activities of LPB CCK neurons at the onset of escape suggest that LPB CCK neurons likely gate and instruct the escape rather than directly control locomotive behaviors.
Despite many early studies mapping downstream projections for LPB neurons (Han et al., 2015a;Saper and Loewy, 1980;Sun et al., 2020b), we are the first to demonstrate that LPB CCK neurons directly connect with PVN neurons. Direct projections from the brainstem to the hypothalamus for the escape responses may be more conducive to escape in emergency situations. Besides the PVN, we also observed that LPB CCK neurons project to the PVT, VMH and PAG, which have also been associated with aversion and/or defensive behaviors (LeDoux, 2012;Vianna et al., 2001;Wang et al., 2015). It would be interesting to further investigate how these different downstream areas coordinately contribute to defensive responses.
PVN is important for neuroendocrine and autonomous regulation (Canteras et al., 2001;Coote, 2005;Ferguson et al., 2008;Sutton et al., 2016). While earlier c-fos staining analyses suggest that PVN neurons are activated in response to different threat stimuli (Canteras et al., 2001;Faturi et al., 2014;Martinez et al., 2008;Staples et al., 2008), it remains unclear whether they are only involved in neuroendocrine responses or also defensive behaviors. Increasing evidence has demonstrated that PVN neurons play important roles in defensive behaviors independent of hormonal actions (Daviu et al., 2020;Mangieri et al., 2019;Xu et al., 2019). Sim1, as a specific marker of PVN, studies have reported that photoactivation of PVN Sim1 neurons induced defensive responses . Our findings support a role for PVN neurons in mediating behavioral responses, as photoactivation of PVN Vglut2 neurons promoted flight-to-nest behavior. Intriguingly, stimulating the LPB C-CK -PVN pathway promoted flight-to-nest behaviors but activating of PVN CRH neurons did not induce apparent flight-to-nest behaviors. Since PVN CRH neurons receive multiple inputs and activation of the PVN CRH neurons have been shown to induce aversion (Kim et al., 2019), future studies using inputspecific activation of PVN CRH neuronal subpopulations would further dissect the role of LPB CCK -PVN CRH pathway in defensive responses. Future studies using additional projection-specific approaches and more genetically defined cell tools may help resolve this problem.
Our data show that activation of LPB CCK neurons drives aversion, defensive and anxiety-like behaviors, evoking an arousal and stressed states. Fear, anxiety and stress responses are closely associated with mental illnesses with dysregulated neural circuits (Blanchard et al., 2001). It is worth further hiding time in the nest (saline: n = 7 mice, CNO: n = 7 mice; for latency, p = 0.0088, t = 3.122, df = 12; unpaired t test; for speed, p < 0.0001, t = 5.736, df = 12; unpaired t test; for time in the nest, p = 0.0032, t = 3.665, df = 12; unpaired t test). (F) Optogenetic activation of LPB CCK -PVN terminals with a cannula implanted in the PVN for aCSF, CNQX +AP5, or Devazepide +L-365 260 delivery. Scale bar, 100 μm. (G) Representative image showing the ChR2-EYFP expression in the LPB of a Cck-cre mouse. Scale bar, 100 μm. (H) Representative image showing the cannula placement in the PVN from a ChR2-EYFP expressing Cck-cre mouse. Scale bar, 100 μm. (I-K) Microinjection of the glutamate receptor antagonists (CNQX +AP5), rather than CCK receptor antagonists (Devazepide +L-365260), into the PVN increased the latency to the nest and reduced the hiding time in the nest (ACSF: n = 6 mice, CNQX +AP5: n = 6 mice, Devazepide +L-365260: n = 5 mice; for latency, F(2,14) = 7.658, p = 0.057; One-way ANOVA; for speed, F(2,14) = 11.49, p = 0.0011; for time in the nest, One-way ANOVA; F(2,14) = 16.44, p = 0.0002; One-way ANOVA). (       Hze /J; Stock No. 007908), C57BL/6 mice and SD rats were obtained from the Shanghai Laboratory Animal Center. Adult male mice and SD rats were used in our study. Mice and rats housed at 22 ± 1 °C and 55 ± 5% humidity on a 12 hr light/12 hr dark cycle (light on from 07:00 to 19:00) with food and water ad libitum. All experimental procedures were approved by the Animal Advisory Committee at Zhejiang University and were performed in strict accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All surgeries were performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

RNAscope in situ hybridization
We used RNAscope multiplex fluorescent reagent kit and designed probes (ACDBio Inc) to perform fluorescence in situ hybridization. Mouse brain tissue was sectioned into 20 μm sections by cryostat (Leica CM 1950). Then sections were mounted on slides and air-dried at room temperature. Subsequently, the sections were dehydrated in 50% EtOH, 70% EtOH, and 100% EtOH for 5-10 min each time and air-dried at room temperature again. Thereafter, protease digestion was performed in a 40 °C HybEZ oven for 30 min pretreatment, slides were hybridized with pre-warmed probe in a 40 °C HybEZ oven for 2 hr. Probes used in our paper were: Slc17a6 probe (VGLUT2, accession number NM_080853.3, probe region 1986-2998), Slc32a1 probe (VGAT, accession number NM_009508.2, probe region 894-2037), Cckar probe (accession number NM_009827.2, probe region 328-1434), Cckbr probe (accession number NM_007627.4, probe region 136-1164). After hybridization, the brain sections went through four steps of signal amplification fluorescent label. Anti-DsRed or GFP staining was performed after the RNAscope staining. Slides were imaged with a confocal microscope (Olympus FluoView FV1200).
For fiber photometry experiments, AAV2/9-hSyn-DIO-GCaMP7s-WPRE virus (viral titers: 2.0×10 12 particles/ml; Taitool Bioscience) were injected into the LPB of CCK-ires-Cre mice. After two weeks, an optical fiber was implanted into the LPB, then each mouse was allowed to recover for 1 week before recording. Each mouse was handled for 3 days prior to fiber photometry recording.

Fiber photometry
Mice were allowed to recover from surgery for at least 7 days before the behavioral experiments. The fiber photometry system (RWD Life Science Co., Ltd, China) was used for recording fluorescence signal (GCaMP7s and isosbestic wavelengths) which produced by an exciting laser beam from 470 nm LED light and 410 nm LED light. Calcium fluorescence signals were acquired at 60 Hz with alternating pluses of 470 nm and 410 nm light. The power at the end of the optical fiber (200 μm, 0.37NA, 2 m) was adjusted to 20 μW. Recording parameters were set based on pilot studies that demonstrated the least amount of photobleaching, while allowing for the sufficient detection of the calcium response. We used the camera for behavioral video recordings to synchronize calcium recordings. On the experimental day, mice were allowed to acclimate in the home cage for 30 min. Regarding quantification, the filtered 410 nm signal was aligned with the 470 nm signal by using the least-square linear fit. ΔF/F was calculated according to (470 nm signal-fitted 410 nm signal)/(fitted 410 nm signal). And the standard z-score calculation method is used, that is, Z-score = (x-mean)/std, x = △F/F. During the behavior experiments, the GCaMP7s fluorescence intensity was recorded.

In vivo optogenetic manipulation
For optogenetic manipulation experiments, an implanted fiber was connected to a 473 nm laser power source (Newdoon Inc, Hangzhou, China). The power of the blue (473 nm; Newdoon Inc, Hangzhou, China) was 0.83-3.33 mW mm 2 as measured at the tip of the fiber. 473 nm laser (ChR2: power 5-15 mW, frequency 20 Hz, pulse width 5ms; GtACR1: power 15 mW, direct current) was supplied to activate or inhibit neurons, respectively.

Pharmacological antagonism
Antagonists were delivered 30 min before optical activation of LPB CCK neurons. For blocking glutamatergic neurotransmission, we firstly connected guiding cannula with a Hamilton syringe via a polyethylene tube. Then we infused 0.25 μl mixed working solution containing CNQX disodium salt hydrate (0.015 μg; Sigma-Aldrich), a glutamate AMPA receptor antagonist, and AP5 (0.03 μg; Sigma-Aldrich), a NMDA receptor antagonist, into the PVN with a manual microinfusion pump (RWD, 68606) over 5 min. For blocking CCKergic neurotransmission, 0.25 μl mixed solution containing Devazepide (0.0625 μg; Sigma-Aldrich), a Cckar receptor antagonist, and L-365,260 (0.0625 μg; Sigma-Aldrich), a Cckbr receptor antagonist, was infused into the PVN over a period of 5 min. To prevent backflow of fluid, the fluid-delivery cannula was left for 10 min after infusion.

c-Fos staining and analysis
For c-fos quantification, mice were perfused 1.5 hr after 10 min blue photostimulation illumination, and sections were cut. The boundaries of the nuclei were defined according to brain atlases Mouse Brain Atlas (Franklin and Paxinos, 2008). Cell counting was carried out manually.

Behavioral task
For all behavioral tests, experimenters were blinded to genotypes and treatments. Mice were handled daily at least seven days before performing behavioral tests. All the apparatuses and cages were sequentially wiped with 70% ethanol and ddH 2 O then air-dried between stages. At the end of behavioral tests, mice were perfused with 4% PFA followed by post hoc analysis to confirm the viral injection sites, optic fiber and cannula locations. Mice with incorrect viral injection sites, incorrect positioning of optical fibers or cannula were excluded.

Real-time place aversion test
Mice were habituated to a custom-made 20×30 × 40 cm two-chamber apparatus (distinct wall colors and stripe patterns) before the test. First stage: each mouse was placed in the center and allowed to explore both chambers without laser stimulation for 10 min. After exploration, the mouse indicated a small preference for one of the two chambers. Second stage: 473 nm laser stimulation (20 Hz, 8 mW, 5ms) was delivered when the mouse entered or stayed in the preferred chamber, and the light was turned off when the mouse moved to the other chamber for 10 min.

Flight-to-nest test
Flight-to-nest test was performed using previously described methods (Zhou, Z., et al, 2019). Flightto-nest test was performed in a 40×40 × 30 cm closed box with a 27-inch LED monitor stationed on top to display the stimulus. A nest in the shape of a 20 cm wide ×12 cm high triangular prism was in the corner of the closed Plexiglas box. There are two cameras, one from the top, one from the side (Logitech) that record the mouse's activity simultaneously. Briefly, on day 1, the mice were habituated to the box conditions for 15 min. On day 2, the mice were first allowed to explore the box for 5-10 min. When the mice were in the corner furthest from the nest and within in a body-length distance from the wall, they were given optical stimulus. For optogenetic activation of LPB CCK neurons or LPB CCK -PVN experiments, mice received a 20 s 473 nm blue laser (frequency 20 Hz, pulse width 5ms) with 15-20 mW (terminal) or 5-10 mW (soma) light power at the fiber tips. For prolonged inhibition of PVN neurons, Clozapine Noxide (CNO, Sigma-Aldrich) was dissolved in saline to a concentration of 3 mg/ml. The flight-to-nest test was performed 1.5 hr after CNO intraperitoneally injection.

Flight-to-nest behavioral analysis
The behaviors of the mice were recorded and analyzed automatically with Anymaze software (Global Biotech Inc). Behavioral analysis was performed as previously described (Zhou, Z., et al, 2019). Flightto-nest behavior was characterized on the basis of the three aspects: latency to return nest, speed (% of baseline speed), time spent in nest (% of 2 min). Latency to return nest refers to the moment from optical stimulus onset to moment when the mouse first went into the nest. Speed (% of baseline speed) refers to the ratio of the post-stimulation speed to the baseline speed. We recorded the speed of the mice in the 50 s before photostimulation presentation as the baseline speed. The post-stimulation speed was record from the time of stimulation to 15 s after. It was averaged over a 1 s time window centered on the maximum speed. Time spent in nest (% of 2 min) refers to the time from mouse's body was first completely under the shelter after photostimulation to the time when the mouse left the nest.

Open-field test
The open-field chamber was made of plastic (50 × 50 × 50 cm). At the start of the test, mice were placed in the periphery of the open-field chamber. The open-field test lasted 5 min.

Elevated plus-maze test
The elevated plus maze was made of plastic with two open arms (30 × 5 cm), two closed arms (30 × 5 × 30 cm) and a central platform (5 × 5 × 5 cm). At the beginning of the experiments, mice were placed in the center platform facing a closed arm. The elevated plus-maze test lasted 5 min.

Heart rate measurements
Heart rate was measured via a pulse oximeter (MouseOx Plus; Starr Life Sciences). Mice were placed in the home cage with a detector fixed around the neck. After habituation for 10 min, heart rate was simultaneously measured for 10 min while intermittent 2 min period blue light was applied. Each mouse was tested three times, and the mean heart rate was calculated. Heart rate data was analyzed with the MouseOx Plus Conscious Applications Software.

Pupil size measurements
Mice were adapted to constant room light (100 lx) for 1 hr before testing. Mice were kept unanaesthetized and restrained in a stereotaxic apparatus during the experiment. The pupil size was recorded using Macro module camera under constant light conditions before stimulation, during stimulation, and after stimulation. The test lasted 90 s, consisting of 30 s light off-on-off epochs. The pupil size was later measured by Matlab software.

Heat exposure assay
We used a translucent plastic box (38×25 cm) divided into two parts, the smaller part is the unheated comfortable zone (5×25 cm), and the larger part with rubber heating pad is the hot uncomfortable zone (33×25 cm). The rubber heating pad was heated to 43℃, and the heat insulated pad was placed in the comfortable zone to avoid the heat. The heating temperature 43℃ was chosen because it would cause heat escape but not heat pain (Wang et al., 2021b;Weisheng et al., 2021).

Rat exposure assay
We used a rectangular chamber (70×25 × 30 cm). Mice were acclimated to this environment for three days for 10 min each day. During the rat exposure period, a live rat was restrained to one end of the chamber using a harness attached to the chamber wall. As a control, before a live rat exposure, we exposed mice to a toy rat during fiber photometry recording (similar in size and shape to a live rat). For fiber photometry recordings, all mice underwent rat exposure for 20 min. For photoinhibition tests, mice were exposed to live rat and all trials lasted 10 min.

Looming test
The looming test was performed in a closed Plexiglas box (40×40 × 30 cm) with a shelter in the corner. For looming stimulation, an LCD monitor was placed on the ceiling to present multiple looming stimuli, which was a black disc expanding from a visual angle of 2° to 20° in 0.5 s. The expanding disc stimulus was repeated for 20 times in quick succession and each repeat is followed by a 0.15 s pause. Animals were habituated for 10-15 min in the looming box one day before testing. During the looming test, mice were first allowed to freely explore for 3-5 min. For calcium signal experiment, total five trials of looming stimuli were presented and analyzed. Behavior was recorded for 20 min. For photoinhibition tests, light stimulation was given 1 s before the looming stimulus appears and continue until the looming stimulus ends.

TMT odor test
During this test, all mice were habituated to the testing environment for three days before any experimental manipulation. For the photometry studies involving odor presentations, mice were placed on a plastic chamber (30×30 × 20 cm). A dish (diameter, 5 cm) with cotton was positioned on the side beside the chamber. When TMT (Ferro Tec, 4 µl of 100%) was used as the stimulus, cotton was first wetted with the equivalent volume of 0.1 M PBS. After a habituation period, the dish was replaced with a new one scented with the TMT. The mice were free to explore in the chamber, and behavior was recorded for 20 min.

Measurements of corticosterone
Blood samples were collected to determine the hormone levels. Taking Blood was performed in the morning by rapidly collecting heart blood after anesthetization. We immediately collected blood after 10 min of light stimulation ( 20 Hz, 5ms pulse width, 15 s per min, 473 nm). Blood samples were temporarily placed in iced plastic tubes coated with heparin. All serum was prepared after every blood sample was centrifuged at 2000 g for 2.5 min at 4 °C. Supernatant was collected and plasma corticosterone concentration was measured using commercially-available ELISA kits (Enzo ADI-900-097).

Quantification and statistical analysis
All data analyses were conducted blinded. All statistical analyses were performed with GraphPad Prism (version 7.0) and analyzed by Unpaired Student's t-tests, one-way ANOVA, two-way ANOVA according to the form of the data. Nonparametric tests were used if the data did not match assumed Gaussian distribution. Animals were randomly assigned to treatment groups. All data were presented as Mean ± SEM, with statistical significance taken as *p<0.05, **p<0.01 and ***p<0.001.

Ethics
Animal experimentation: All experimental procedures were approved by the Animal Advisory Committee at Zhejiang University and were performed in strict accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Laboratory Animal Welfare and Ethics Committee of Zhejiang University; National Institutes of Health Guidelines for the Care and Use of Laboratory Animals; AP CODE:ZJU20220160.

Supplementary files • MDAR checklist
Data availability All data generated or analysed during this study are included in the manuscript and supporting file. Source data files have been uploaed.